Stabilized enzymatic antimicrobial compositions

ABSTRACT

A stabilized aqueous composition capable of producing or, in the presence of saliva or other humoral fluid, leading to the production of antimicrobially effective concentrations of hypothiocyanite ions (OSCN--) are herein described. The composition contains an oxidoreductase enzyme and its specific substrate, for the purpose of producing hydrogen peroxide of at least the minimum effective concentration, and in addition, catalase for the destruction of hydrogen peroxide to prevent premature oxidoreductase enzyme decomposition. Optionally, a peroxidase enzyme may be included to act upon the aforementioned hydrogen peroxide, thereby oxidizing thiocyanate ions to produce the antimicrobial concentrations of hypothiocyanite ions (OSCN--).

STATEMENT OF RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.07/797,776 filed on Nov. 25, 1991, now U.S. Pat. No. 5,176,899 issued onJan. 5, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to antimicrobial compositions which are capableof activating or supplementing naturally occurring peroxidase systems.In particular, compositions which are stable during preparation andafter packaging in environments which contain some oxygen, methods ofmaking such compositions, and methods of use are disclosed.

2. Art Background

There is general acceptance as to the etiology of dental caries andperiodontal disease in that microflora found in the oral environment arecapable of accumulating upon oral surfaces and in unexposed pockets,thriving and producing damaging metabolites in the absence of properdental hygiene. Colonies of microbes, undisturbed for even short periodsof time, are able to aggressively adhere to the surface of enamel,establishing a foothold for further colony growth. Many of the bacterialtypes commonly found in the mouth secrete polysaccharides such asglucans and dextrans, which form a supportive matrix and thus provide amore mechanically stable environment for further proliferation.Subgingivally, undisturbed colonies of aerobic and anaerobic bacteriacan establish similar polysaccharide matrices, in addition topocket-type formations.

These polysaccharide matrices, together with the thriving microfloracontained therein, make up what is commonly referred to as plaque. Thefirst stages in plaque formation occur almost immediately after anenamel surface is scraped, cleaned and polished in dental office toothcleaning procedures. As colony numbers increase, and the structuralintegrity of the surrounding polysaccharide matrix evolves, plaquebecomes a potential source of bacterial metabolites such as lactic acid.In intimate contact with the enamel surface, acidic plaque metabolitesare thus capable of lowering the pH of the enamel surface to a point atwhich demineralization of the hydroxyapatite can occur. Suchdemineralization is known to be the cause of tooth decay, also known ascaries. Subgingivally, plaque and pocket colonies are known to causedemineralization of both enamel and periodontal bone structure.Gingivitis and periodontitis, infection and irritation of the softtissues surrounding the teeth, are other clinical manifestations ofsubgingival plaque and pocket colony proliferation.

One approach taken to decrease caries is by limiting thedemineralization of enamel and bone through drinking water fluoridation.It has been shown that the fluoride provided by drinking water (and to amore limited extent, through diet) is capable of being incorporated intohydroxyapatite, the major inorganic component of enamel and bone.Fluoridated hydroxyapatite is less susceptible to demineralization byacids and is thus seen to resist the degradative forces of acidic plaqueand pocket metabolites. In addition, fluoride ion concentration insaliva is increased through consumption of fluoridated drinking water.Saliva thus serves as an additional fluoride ion reservoir; incombination with buffering salts naturally found in salivary fluid,fluoride ions are actively exchanged on the enamel surface, furtheroffsetting the effects of demineralizing acid metabolites.

A large body of data indicates that drinking water fluoridation leads toa statistically significant decrease in DMF (decayed, missing, andfilled) teeth for a broad range of populations studied. Smaller, lesssignificant effects are seen in fluoridated drinking water studies whichexamine changes in periodontal health. Positive periodontal effects arethought to arise through the antimicrobial effects of increased fluorideion concentration in saliva.

However, notwithstanding the established benefits of fluoride treatmentof teeth, fluoride ion treatment can result in the mottling of teeth,whether administered systemically through drinking water or topicallyapplied. This effect is known to be both concentration related andpatient-specific. In addition, the toxicology of fluoride has recentlycome under closer scrutiny, although there is no clear answer as to itslong term effect on human health. However, for the time being, drinkingwater fluoridation is believed to serve a wider public good, and itseffect on the dental health of populations the world over arepronounced.

Another approach to limiting the proliferation of microflora in the oralenvironment is through the topical or systemic application ofbroad-spectrum antibacterial compounds. By killing large numbers of oralmicroflora, it is postulated, plaque and pocket accumulation, togetherwith their damaging acidic metabolite production, can be reduced oreliminated. The major drawback to such an approach is that there are awide variety of benign or beneficial strains of bacteria found in theoral environment, which are killed by the same antibacterial compoundsin the same manner as the harmful strains. In addition, such treatmentwith antibacterial compounds may select for certain bacteria and mostfungi, which may then be resistant to the antibacterial compoundadministered, and thus proliferate, unrestrained by the symbiotic forcesof a properly balanced microflora population. Such a selectedproliferative process leads to yet another clinical problem which mustthen be addressed with other antimicrobial strategies. Thus, theapplication or administration of broad-spectrum antibiotics isill-advised, except in preventative or palliative clinical situationssuch as oral surgery, severe periodontitis, and immune dysfunctiondiseases.

Less potent and more selective antimicrobial compounds have beendevised, which, when applied topically, have achieved varying degrees ofsuccess in checking the growth of harmful oral microorganisms. Ofparticular interest and relevance to the subject matter of the presentinvention are those approaches which attempt to activate or supplementthe antimicrobial potential of saliva.

Saliva is known to contain a variety of immunoglobulin andnon-immunoglobulin antibacterial compounds as a defense against theproliferation of harmful pathogens. Such non-immunoglobulin proteinsinclude lysozyme, lactoferrin and salivary peroxidase. These proteins,or ones similar in function, are found in virtually all mammalianmucosal secretions, providing a first line of defense against pathogenicorganisms which would otherwise rapidly proliferate in such warm, moistenvironments. The enzyme salivary peroxidase, or SPO, functions byutilizing hydrogen peroxide (produced and excreted primarily by certainbacteria as a metabolite, but found also in newly expressed saliva) tooxidize a pseudohalide ion found in saliva, thiocyanate (SCN--), toproduce a potent bacteriostatic agent, hypothiocyanite ion (OSCN--).Hypothiocyanite ion and its corresponding acid, hypothiocyanous acid(herein referred to collectively as hypothiocyanite) are able to inhibitthe growth of a wide variety of harmful pathogens found in the oralenvironment. Depending upon the concentration of hypothiocyanite in thesaliva, the salivary peroxidase system can either merely inhibitmicrobial metabolism or actually kill the organism. In general, it hasbeen shown that concentrations of hypothiocyanite greater than about 100micromoles/liter are sufficient to inhibit the metabolism of plaquebacteria.

Since the salivary peroxidase system, and thus the production ofhypothiocyanite, is dependent upon the availability of hydrogenperoxide, various prior art attempts to provide sufficient hydrogenperoxide to activate or supplement the SPO system have been made.Conversely, since SPO begins to show inhibition by concentrations ofhydrogen peroxide greater than about 1 millimole/liter, an effective SPOactivation mechanism should not provide or accumulate peroxidemolarities much higher than this. Direct inclusion of hydrogen peroxidein a mouth rinse composition at these low concentrations has been shownto activate the SPO system for short periods of time(Mansson-Rahemtulla, et al., J. of Dental Res. 62(10): 1062-1066).Another prior art attempt to generate hydrogen peroxide in situcomprised including an oxidoreductase enzyme, such as glucose oxidase,in a dentifrice (Hoogendorn, et al., U.S. Pat. Nos. 4,150,113 and4,178,519). The glucose oxidase thus provided would, upon oralapplication, react with glucose present in saliva and in plaqueinterstitial fluid to produce hydrogen peroxide at low concentrations.Since this approach was dependent upon the availability of glucose inthe mouth, a more reproducible and predictable route to enzymatichydrogen peroxide production was then taken by the present inventor andothers by including both glucose oxidase and beta-D-glucose within adentifrice composition. (U.S. Pat. No. 4,537,764). Beta-D-glucose is theanomer of glucose for which glucose oxidase is specific; in aqueoussolution, glucose will mutorotate rapidly to form a mixture ofapproximately 65% beta-D-glucose and 35% alpha-D-glucose. In order toprevent instability and premature enzyme/substrate interaction theamount of water in the composition had to be limited to less than 10percent. Upon use of this dentifrice composition, additional waterpresent (from saliva and from water added in the course of normaltoothbrushing procedures) would dilute the composition to a watercontent of greater than 10 percent, thus allowing reaction betweenglucose oxidase and glucose to ensue. The hydrogen peroxide thus createdas a product of reaction would activate the salivary peroxidase systemin saliva, producing hypothiocyanite.

Later attempts were made to provide a dentifrice composition containinga complete system of components capable of generating hypothiocyanite insitu (U.S. Pat. Nos. 4,564,519 and 4,578,265). An oxidoreductase enzymetogether with its corresponding substrate were combined in a singledentifrice composition with a peroxidase enzyme and a thiocyanate salt,thus providing a method of producing hypothiocyanite independent offluctuations in salivary glucose, salivary peroxidase and salivarythiocyanate ion. Again, stability of such dentifrice compositionscontaining a complete enzymatic system capable of producinghypothiocyanite could only be maintained by formulating with less thanabout 10 percent water. Similarly, the reaction sequence was started bydilution of the dentifrice during toothbrushing.

There are numerous other examples in the prior art of attempts toprovide a stable enzymatic dentifrice containing both an oxidoreductaseenzyme and its specific substrate for the purpose of producing hydrogenperoxide. Stability of such prior art compositions has been achieved byeither limitations placed on the amount of water contained within thecomposition or by physically separating (through microencapsulation,U.S. Pat. No. 4,978,528) the oxidoreductase enzyme from its specificsubstrate.

In light of the foregoing description, it would be advantageous toprovide a stable, aqueous enzymatic composition capable ofsupplementing, or, in the presence of saliva or other humoralsecretions, activating the peroxidase system in such a fashion thathypothiocyanite ions (OSCN--) are produced in excess of about 100micromoles/liter/minute in vitro or in vivo.

In addition to the salivary peroxidase system, other naturalantimicrobial systems can be found in the humoral secretions of allmammals. These antimicrobial systems consist of a peroxidase enzyme (forexample, salivary peroxidase found in saliva and cervical peroxidasefound in vertical fluid) together with an oxygen acceptor such asthiocyanate or iodide ion. Both components are normally found in thesecretions themselves, and the addition of exogenous hydrogen peroxideactivates the system and produces potent antimicrobial species, such ashypothiocyanite and hypoiodite (OSCN-- and OI--, respectively).

As described in my prior application referenced above, aqueouscompositions capable of producing hydrogen peroxide enzymaticallythrough the action of an oxidoreductase enzyme must be manufactured soas to contain a low level of dissolved oxygen in order to prevent thepremature formation of hydrogen peroxide prior to the time of intendeduse. Such compositions must also be packaged in an essentially anaerobicfashion so as to exclude oxygen during storage. These requirements arethe subject of my prior application referred to above.

It would also be advantageous to provide a stable, aqueous enzymaticcomposition capable of producing or, in the presence of saliva, leadingto the production of hypothiocyanite, irrespective of the composition'swater content or the amount of water available for dilution upon use.Additionally, formulating latitude and economy would greatly benefitfrom such aqueous enzymatic dentifrice compositions produced andstabilized without regard to the amount of water contained within theformulation. It would also be advantageous to provide such a compositionwhich may be formulated in the presence of oxygen without having to beparticularly careful about removing and/or replacing the oxygen which ispresent until it is packaged.

It would be of additional utility to provide a method of manufacturing astable, aqueous enzymatic composition which contains both anoxidoreductase enzyme and its specific substrate, yet prevents hydrogenperoxide accumulation prior to its intended use.

SUMMARY OF THE INVENTION

In accordance with the foregoing description of the prior art and adesire to provide a stabilized aqueous composition capable of producingor, in the presence of saliva, leading to the production ofantimicrobial concentrations of hypothiocyanite ions (OSCN--),compositions are herein described which contain both an oxidoreductaseenzyme and its specific substrate, for the purpose of producing hydrogenperoxide of at least the minimum effective concentration. The aqueousdentifrice compositions of the present invention can be stabilizedagainst premature enzyme/substrate interaction by controlling the levelof dissolved oxygen in the aqueous dentifrice carrier. Optionally, aperoxidase enzyme may be included to act upon the aforementionedhydrogen peroxide, thereby oxidizing thiocyanate ions (found in salivaor optionally included in the present compositions) to produce theantimicrobial concentrations of hypothiocyanite ions (OSCN--).

In the case where the present invention is used as a dentifricecomposition, in addition to containing ingredients normally found indentifrice compositions and well known to those familiar with the art,the aqueous dentifrice compositions of the invention also contain anoxidoreductase enzyme, together with said enzyme's specific substrate,in sufficient quantities to produce hydrogen peroxide at a rate of fromabout 0.1 millimoles/liter/minute to about 10.0 millimoles/liter/minuteduring use. The reaction sequence is, in general, as follows: ##STR1##

In addition, the aforementioned compositions may contain a peroxidaseenzyme capable of acting upon the enzymatically produced hydrogenperoxide and thereby oxidizing thiocyanate ions (normally found insaliva) to form hypothiocyanite ions (OSCN--). The level of peroxidaseenzyme in such compositions shall in the preferred embodiment, besufficient to, when in contact with saliva (which contains thiocyanateions), cause the production of at least 100 micromoles/liter/minute ofhypothiocyanite ions (OSCN--) during use. Optionally, thiocyanate ionsmay also be included in the compositions of this invention in an amountsufficient, together with the other inventive ingredients, to produce inexcess of about 100 micromoles /liter/minute of hypothiocyanite ionsduring use. The reaction sequence is, in general, as follows: ##STR2##

Another aspect of the present invention is that it has been discoveredthat, irrespective of the amount of water contained in dentifricecompositions comprising an oxidoreductase enzyme together with itsspecific substrate, premature enzyme/substrate interaction can beeliminated by limiting the amount of dissolved oxygen in the aqueousdentifrice carrier. Thus it is possible to provide an aqueous dentifricecomposition containing both an oxidoreductase enzyme and its specificsubstrate, for the purpose of producing hydrogen peroxide upon use,which will show little or no hydrogen peroxide accumulation in advanceof its intended utility. Only upon exposure to additional oxygen willthe dentifrice compositions of the present invention be shown to beenzymatically active. The manipulation and control of dissolved oxygenlevels in enzymatic dentifrice compositions, for the purpose of limitingan oxidoreductase enzyme/substrate interaction and thereby stabilizingsaid enzymatic dentifrice until its intended time of use, is unknown inthe prior art.

An unexpected benefit of the ability to control the interaction betweenthe oxidoreductase enzyme and its specific substrate by controlling theamount of dissolved oxygen in the aqueous dentifrice carrier, is thepossibility of turning the reaction "on" and "off" at any given time inthe course of its manufacture. Thus, controlled concentrations ofhydrogen peroxide (or alternatively, if additionally formulated withboth a peroxidase and thiocyanate ions, hypothiocyanite ions) may beproduced in the course of manufacturing in order to reduce any microbialpopulations without the use of preservatives.

It has been found that the addition or inclusion of catalase (EC1.11.1.6) in compositions capable of producing hydrogen peroxideenzymatically (such as glucose oxidase and glucose) greatly improves theretention of oxidoreductase activity over the course of manufacturing,packaging and storing the composition. At the point of use, the hydrogenperoxide produced is then consumed almost exclusively by theantimicrobial peroxidases, due to their greater affinity for hydrogenperoxide than catalase.

In general, the ratio of oxidoreductase enzyme to catalase should be inthe range of 50 Titrimetric Units of oxidoreductase to 1.0 Baker Unit ofcatalase, to 2.0 Titrimetric Units of oxidoreductase to 1.0 Baker Unitof catalase (50:1 to 1:1).

One Titrimetric Unit (TU) is defined herein as that amount ofoxidoreductase enzyme capable of producing 1.1 micromole of hydrogenperoxide per minute at a temperature of 35° C., and optimal conditionsof pH and substrate concentration for each particular oxidoreductaseenzyme.

One Baker Unit (BU) is equivalent to that amount of catalase capable ofdecomposing 264 mg of hydrogen peroxide in one hour under the conditionsof the assay (25° C. and pH 7.0)

DETAILED DESCRIPTION OF THE INVENTION

In its simplest form, this invention comprises aqueous compositionscontaining an oxidoreductase enzyme and a substrate specific to saidenzyme for the purpose of producing hydrogen peroxide upon use incombination with catalase for decomposing hydrogen peroxide in thecomposition. In particular, only those oxidoreductase enzymes whichutilize water as a co-reactant and oxygen as an electron donor, therebyproducing hydrogen peroxide upon reaction with a specific substrate, arecontemplated.

Suitable oxidoreductases include, but are not limited to, glucoseoxidase, galactose oxidase, glycollate oxidase, lactate oxidase,L-gulunolactone oxidase, L-2-hydroxyacid oxidase, aldehyde oxidase,xanthine oxidase, D-aspartate oxidase, L-amino acid oxidase, D-aminoacid oxidase, monoamine oxidase, pyridoxaminephosphate oxidase, diamineoxidase, and sulfite oxidase. The preferred oxidoreductase is glucoseoxidase.

Suitable substrates are specific to the particular oxidoreductase chosenand are well known in the art. For instance, beta-D-glucose is aspecific substrate for glucose oxidase. Other suitable substratesinclude, but are not limited to D-glucose, D-galactose, L-sorbose,ethanol, tyramine, 1, 4-diaminobutane, 6-hydroxy-L-nicotine,6-hydroxy-D-nicotine, 2-aminophenol, glycollate, L-lactate,2-deoxy-D-Glucose, L-gulunolactone, L-galactonolactone,D-mannonolactone, L-2-hydroxyisocaproate, acetaldehyde, butyraldehyde,xanthine, D-aspartate, D-glutamate, L-amino acids and D-amino acids.

The inventive dentifrice compositions shall thus contain at least one ofthe above oxidoreductases and at least one substrate specific to saidoxidoreductase, for the purpose of producing hydrogen peroxide at a rateof from about 0.10 millimoles/liter/minute to about 10.00millimoles/liter/minute during use. Hydrogen peroxide production may becontrolled by varying either the concentration of oxidoreductase or theconcentration of substrate. For a given rate of hydrogen peroxideproduction, it is seen to be more economical to increase the level ofsubstrate in the dentifrice composition in order to maximize the rateachievable at a specific oxidoreductase level. Oxidoreductaseconcentrations may be subsequently increased if substrate enhancement nolonger yields a higher or desired rate of hydrogen peroxide production.In general, substrate concentrations may range from about 0.01 percentto about 20 percent or more, by weight of the dentifrice composition.

The compositions of the present invention contain, in general from about0.10 Titrimetric Units to about 100 Titrimetric Units of anoxidoreductase enzyme per gram of dentifrice.

The hydrogen peroxide producing dentifrice described above mayoptionally include a peroxidase enzyme for the purpose of utilizing saidhydrogen peroxide to oxidize thiocyanate ions (SCN--), which arenormally found in saliva, to antimicrobial hypothiocyanite ions(OSCN--). Any peroxidase capable of utilizing hydrogen peroxide tooxidize thiocyanate is contemplated to have utility in the practice ofthis portion of the invention.

Suitable peroxidases include, but are not limited to, lactoperoxidase,myeloperoxidase, salivary peroxidase, and chloroperoxidase. Thepreferred peroxidase is lactoperoxidase.

The concentration of peroxidase shall be sufficient to producehypothiocyanite ions at a rate of about 100 micromoles/liter/minute wheninteracting with the hydrogen peroxide produced by theoxidoreductase/substrate reaction and the thiocyanate ions found insaliva. In general, the compositions of this invention may contain from0.10 ABTS Units to about 1000 ABTS Units of peroxidase per gram ofdentifrice.

One ABTS Unit, for the purpose herein, is the amount of peroxidasecapable of oxidizing one micromole of ABTS(2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) per minute at25° C., 0.1M phosphate buffer (pH 6.0), and initial hydrogen peroxideconcentration of 0.100M.

Optionally, the dentifrice compositions described above may contain athiocyanate ion source in order to provide a complete hypothiocyaniteion producing dentifrice, independent of the availability of such ionsin saliva. Thiocyanate ions may be included in the composition atconcentrations of from about 0.10 millimoles/gram of dentifrice to about10.00 millimoles/gram of dentifrice. Thiocyanate ion sources such assodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, andother thiocyanate salts are contemplated to have utility in suchcomplete systems. The preferred thiocyanate ion sources are potassiumthiocyanate and sodium thiocyanate.

In addition to the inventive ingredients described above, the dentifricecompositions of this invention are seen to contain many of thecomponents normally found in such compositions and readily familiar tothose skilled in the art. A number of ingredients in the inventivecompositions are included or chosen to address the stabilityrequirements for the oxidoreductase and, optionally, the peroxidaseenzymes.

Dentifrice compositions of this invention contain a fluid carrier,comprised of water and a humectant, in an amount ranging from about 10%to about 90% by weight of the composition. Suitable humectants include,but are not limited to glycerine, propylene glycol, sorbitol (70%solution), polyethylene glycols, polypropylene glycols, and mixturesthereof. The water content of the composition may typically range fromabout 5% to about 50% by weight, most preferably from about 10% to about30% by weight of the total composition.

In order to achieve the desirable aesthetics of flow and flavor releasein the final dentifrice composition, a thickener is advantageouslyincluded to provide and control viscosity and thixotropy. Suitablethickeners include natural and synthetic water-soluble polymers such assodium carboxymethylcellulose, xanthan gum, carrageenan, locust beangum, gum tragacanth, hydroxyethylcellulose (Natrosol, Hercules, Inc.),sodium alginate, starch, polyvinylpyrrolidone, polyacrylic acid(Carbopol, B. F. Goodrich), and others. Inorganic thickeners such asmagnesium aluminum silicate (Veegum, R. T. Vanderbilt), hectorites (suchas Laponite, La Porte, Ltd.), and hydrated silicas (Sylodent, W. R.Grace), among others, are also useful thickeners for the dentifricecompositions of this invention.

The removal of plaque and tartar by the physical motion of toothbrushingis improved by the inclusion of abrasives in the dentifrice composition.Abrasives commonly included in typical dentifrice compositions arecontemplated and include, but are not limited to, calcium pyrophosphate,calcium carbonate, hydrated silica (Sylodent), aluminum hydroxide,dicalcium phosphate dihydrate, tricalcium phosphate, sodiummetaphosphate, potassium metaphosphate, aluminum silicate, finelydivided poly(methyl methacrylate), and mixtures thereof. In general asis known in the art, the abrasive is present in the composition inconcentrations of from about 5% to about 70% by weight, and preferablyfrom about 10% to about 50% by weight of the total composition. Adentifrice's degree of abrasivity can be measured directly or estimatedfrom the RDA (Radioactive Dentin Abrasion) scale. The RDA scale is ameasure of an abrasive's ability to erode the surface of enamel afterrepeated brushing. The higher the RDA score, the more enamel abradedunder given conditions. High RDA scores are desired for stain removingdentifrices; low RDA scores are desired for dentifrices for sensitiveteeth.

Dentifrices generally contain a foaming agent, or surfactant, to achievethe desired body and texture during toothbrushing. In addition, thesurfactant provides a positive psychological impression of the cleansingprocess, and, to a lesser degree, helps to soften food particles andplaque to assist in their removal by mechanical means. Althoughdesirable, it is by no means necessary to include a surfactant in thedentifrice compositions of this invention. In fact, dentifricecompositions which are not intended to be rinsed following thetoothbrushing procedure, such as compositions utilized in veterinarydentistry or oral care products for individuals unable to brush theirteeth by normal means, should not contain ingredients, includingsurfactants, which are not intended or acceptable for ingestion. Inthose compositions where the presence of a surfactant is desirable,though, compatibility of the surfactant with the enzyme or enzymes ofthe inventive compositions must be confirmed. Many, but not all, anionicsurfactants, such as sodium lauryl sulfate (a commonly employed foamingagent for dentifrice compositions), are known to complex with andinactivate a wide variety of enzymes. Many cationic surfactants are alsoincompatible with enzymes. In general, nonionic and amphotericsurfactants are preferred in the present dentifrice compositions, asthey exhibit, on the whole, much better overall compatibility withenzymes. The prior art addresses the problem of enzyme/surfactantincompatibility at length, and the compatibility of a particularsurfactant with the inventive dentrifice compositions must be determinedon an individual, compound by compound basis. Surfactants known to becompatible with the enzymatic dentifrice compositions of this inventioninclude, but are not limited to, polysorbate 80, cocoamidopropylbetaine,cocoamphopropionate, sodium lauroyl sarcosinate, ethoxylated (20)isocetyl alcohol, and a wide variety of propylene oxide/ethylene oxideblock copolymer nonionic surfactants, such as those offered under thePluronic tradename by BASF/Wyandotte Corp.

Since enzymes are more stable and show higher activity at specific pHlevels, it is advantageous to provide one or more buffering compounds inthe enzymatic dentifrice compositions. Buffers which provide adentifrice and/or in-use pH of approximately 5.5 to 7.5 are seen to bemost beneficial in optimizing the levels of hydrogen peroxide and/orhypothiocyanite ions produced. Any physiologically acceptable bufferproviding a dentifrice and/or in-use pH value of from about 5.5 to about7.5, and preferably between pH 6.0 and pH 7.0, is anticipated havingutility in the practice of this invention. The preferred buffers arepotassium phosphate, sodium phosphate, disodium phosphate, dipotassiumphosphate, and mixtures thereof. The preferred buffer concentrations arefrom about 0.01 moles to about 1.00 moles/liter of fluid dentifricecarrier (that part of the dentifrice excluding insoluble components suchas abrasives).

A wide variety of auxiliary dentifrice components may be included in thepresent compositions, such as preservatives, whiteners, dyes, fluorides,antitartar and anticalculus agents, chlorophyll compounds, ammoniatedmaterials, and others. Such auxiliary components should be compatiblewith the components and desired purpose of the enzyme/substrate systemof the invention.

A suitable flavoring and/or sweetening material may be employed toachieve the desired aesthetics for the dentifrice. Examples of suitableflavoring components are oils of peppermint, spearmint, clove,wintergreen, cinnamon, sage, eucalyptus and orange. Suitable sweeteningagents include saccharin, sodium cyclamate, aspartyl phenylalanine(methyl ester), glucose, xylitol, sucrose, maltose, and others.Flavoring and sweetening agents may comprise from about 0.1% to about7.0% or more of the dentifrice composition.

As a practical matter, manufacturing should be carried out in alow-oxygen environment, such as a vacuum, or under a nitrogen gasblanket, although such steps are not required.

The dentifrice compositions of this invention are intended to be used orotherwise applied in the manner of normal toothbrushing. Residence orcontact time in the oral environment should be at least 30 seconds andpreferably from about 60 seconds to 120 seconds or longer. Normally, thedentifrice is rinsed from the mouth following toothbrushing, however,non-rinse or ingestible compositions are anticipated to have utility aspreviously discussed.

The activities of enzymes are generally measured in terms of micromolesof substrate or co-reactant consumed, or micromoles of product produced,over a given period of time, under specific conditions of temperature,substrate concentration, and co-reactant concentrations. Any descriptionof "unit" activity for a given enzyme should be considered carefully byevaluating a complete description of the conditions under which suchactivity was measured, and the present invention, as defined by theclaims, is considered to be of appropriate scope to encompass thebroadest definition of the term.

In light of the aforementioned definitions of Unit activity foroxidoreductases and peroxidases, the compositions of the presentinvention contain, in general from about 0.10 Titrimetric Units to about100 Titrimetric Units of an oxidoreductase enzyme per gram ofdentifrice, and, optionally, from about 0.10 ABTS Units to about 1,000ABTS Units of a peroxidase enzyme per gram of dentifrice.

An example of such a composition is a toothpaste which contains glucoseand glucose oxidase for the purpose of producing hydrogen peroxide uponuse. The inclusion of catalase in example 2 below demonstrates improvedstability compared to Example 1. Both Examples 1 and 2 were manufacturedunder a partial vacuum (20" Hg) and packaged in foil/plastic laminatedtoothpaste tubes.

EXAMPLE 1

    ______________________________________                                        INGREDIENT         PERCENTAGE/AMOUNT                                          ______________________________________                                        Sorbitol 70%       37.44%                                                     Glycerin 96%       16.00                                                      Dionized water     16.77                                                      Sodium carboxymethylcellulose                                                                     1.00                                                      9M31XF                                                                        Sylodent 750       12.00                                                      Sylodent 2         12.00                                                      Dextrose            2.00                                                      Titanium dioxide    0.40                                                      Other ingredient    2.39                                                      Glucose oxidase    6.0 TU/gm paste                                            Catalase           0.06 BU/gm paste                                           ______________________________________                                    

EXAMPLE 2

    ______________________________________                                        INGREDIENT         PERCENTAGE/AMOUNT                                          ______________________________________                                        Sorbitol 70%       37.44%                                                     Glycerin 96%       16.00                                                      Dionized water     16.77                                                      Sodium carboxymethylcellulose                                                                     1.00                                                      9M31XF                                                                        Sylodent 750       12.00                                                      Sylodent 2         12.00                                                      Dextrose            2.00                                                      Titanium dioxide    0.40                                                      Other ingredients   2.39                                                      Glucose oxidase    6.0 TU/gm paste                                            Catalase           1.0 BU/gm paste                                            ______________________________________                                    

Example 2 retained over 95% of its original glucose oxidase activitywhen stored at 35° C. for 14 days, while Example 1 retained less than 8%of its original activity under the same conditions.

The foregoing description of the invention is intended to be exemplarywith respect to certain preferred embodiments and it will be understoodthat modifications and variations thereof obvious to those skilled inthe art are to be included within the scope of this application and theappended claims.

What is claimed is:
 1. An antimicrobial dentifrice composition made bythe process comprising the steps of:providing a fluid carrier comprisingan oxidoreductase enzyme, an oxidoreductase enzyme substrate and acatalase, wherein said enzyme and substrate form hydrogen peroxide whenreacted together in the presence of oxygen, said hydrogen peroxide beingformed at a rate of at least 100 micromoles per liter per minute, saidoxidoreductase enzyme being present in said composition in the amount ofat least 1.0 Tritrmetric Unit per gram of dentifrice, and wherein saidcatalase is provided in a ratio of about 50 Titrimetric Unitsoxidoreductase enzyme to 1.0 Baker Unit of catalase, to 1.0 TitrimetricUnits oxidoreductase enzyme to 1.0 Baker Unit of catalase tosubstantially minimize the amount of hydrogen peroxide produced in saidcomposition during storage; and storing said mixture in an oxygenimpervious container.
 2. The composition of claim 1 wherein catalase isprovided in an amount in the ratio range of 50 Titrimetric Units ofoxidoreductase to 1.0 Baker Unit of catalase, to 1.0 Titrimetric Unitsof oxidoreductase to 1.0 Baker Unit of catalase.
 3. The composition ofclaim 2 wherein the ratio of oxidoreductase to catalase is about 6.0Titrimetric Units of oxidoreductase to 1.0 Baker Unit of catalase. 4.The composition of claim 1 further comprising a peroxidase enzyme foroxidizing thiocyanate ions to hypothiocyanite ions.
 5. The compositionof claim 1 wherein said oxidoreductase is selected from the groupconsisting of glucose oxidase, galactose oxidase, glycollate oxidase,lactate oxidase, L-gulunolactone oxidase, L-2-hydroxyacid oxidase,aldehyde oxidase, xanthine oxidase, D-aspartate oxidase, L-amino acidoxidase, D-amino acid oxidase, monoamine oxidase, pyridoxaminephosphateoxidase, diamine oxidase, and sulfite oxidase.
 6. The composition ofclaim 1 wherein said oxidoreductase is glucose oxidase.
 7. Thecomposition of claim 1 wherein said substrates are specific to theparticular oxidoreductase and are selected from D-glucose, D-galactose,L-sorbose, ethanol, tyramine, 1, 4-diaminobutane, 6-hydroxy-L-nicotine,6-hydroxy-D-nicotine, 2-aminophenol, glycollate, L-lactate,2'-deoxy-D-Glucose, L-gulunolactone, L-galactonolactone,D-mannonolactone, L-2-hydroxyisocaproate, acetaldehyde, butyraldehyde,xanthine, D-aspartate, D-glutamate, L-amino acids and D-amino acids. 8.The composition of claim 1 wherein said oxidoreductase is glucoseoxidase and said substrate is D-glucose.
 9. The composition of claim 3wherein the peroxidase enzyme is selected from lactoperoxidase,myeloperoxidase, salivary peroxidase, and chloroperoxidase.
 10. Thecomposition of claim 9 wherein the peroxidase is lactoperoxidase. 11.The composition of claim 1 wherein the catalase is derived from A. nigerfermentation.
 12. The composition of claim 1, wherein said compositioncomprises a fluid carrier, comprised of water, in an amount ranging fromabout 10% to about 90% by weight of the composition.
 13. The compositionof claim 1 further comprising a humectant selected from glycerine,propylene glycol, sorbitol (70% solution), polyethylene glycols,polypropylene glycols, and mixtures thereof.
 14. The composition ofclaim 12 wherein said water comprises in the range from about 5% toabout 50% by weight of said composition.
 15. The composition of claim 1further comprising a thickener selected from natural and syntheticwater-soluble polymers selected from sodium carboxymethylcellulose,xanthan gum, carrageenan, locust bean gum, gum tragacanth,hydroxyethylcellulose, sodium alginate, starch, polyvinylpyrrolidone andpolyacrylic acid and inorganic thickeners selected from magnesiumaluminum silicate, hectorites and hydrated silicas.
 16. The compositionof claim 1 further comprising abrasives selected from the groupconsisting of calcium pyrophosphate, calcium carbonate, hydrated silica,aluminum hydroxide, dicalcium phosphate dihydrate, tricalcium phosphate,sodium metaphosphate, potassium metaphosphate, aluminum silicate, finelydivided poly(methyl methacrylate), and mixtures thereof.
 17. Thecomposition of claim 1 wherein said composition further comprises aphysiologically acceptable buffer.
 18. The composition of claim 17wherein said physiologically acceptable buffer is selected frompotassium phosphate, sodium phosphate, disodium phosphate, dipotassiumphosphate, and mixtures thereof.
 19. The composition of claim 1 furthercomprising additives selected from the group comprising preservatives,whiteners, dyes, fluorides, antitartar and anticalculus agents,chlorophyll compounds, ammoniated materials, flavorings and sweeteners.20. Method of making a dentifrice composition comprising the stepsof:mixing together an oxidoreductase enzyme, an oxidoreductase enzymesubstrate and a catalase, wherein said enzyme and substrate formhydrogen peroxide when reacted together, said hydrogen peroxide beingformed at a rate of at least 100 micromoles per liter per minute saidoxidoreductase enzyme being present in said composition in the amount ofat least 1.0 Titrimetric Unit per gram of dentifrice and wherein saidoxidoreductase enzyme and catalase are present in a fluid carrier in aratio of about 50 Titrimetric Units oxidoreductase enzyme to 1.0 BakerUnit of catalase, to 1.0 Titrimetric Units oxidoreductase enzyme to 1.0Baker Unit of catalase; and storing said mixture in an oxygen imperviouscontainer.
 21. The method of claim 20 wherein the composition is madeeither under a partial vacuum or in an oxygen free atmosphere after saidoxidoreductase enzyme and an oxidoreductase enzyme substrate are addedthereto.
 22. The method claim 21 wherein said step of limiting theamount of oxygen in said composition is performed under an inert gas.23. An anaerobically packaged composition with an antimicrobial systemcomprising:a fluid carrier comprising an oxidoreductase enzyme and anoxidoreductase enzyme substrate, wherein said enzyme and substrate formhydrogen peroxide when reacted together in the presence of oxygen, saidhydrogen peroxide is formed at a rate of at least 100 micromoles perliter per minute; and catalase provided in sufficient amount tosubstantially reduce the amount of hydrogen peroxide in saidcomposition; said composition being packaged in an oxygen imperviouspackage or container.